Enhancement of enzyme reactions

ABSTRACT

This invention relates to activation of enzymes. More specifically, the invention relates to agents capable of enhancing the activity of peroxidases or peroxidase acting compounds. The invention also relates to methods of oxidizing a substrate with a source of hydrogen peroxide in the presence of a peroxidase enzyme or a peroxidase acting compound, and an enhancing agent. More specifically, the invention relates to a method of bleaching of dye in solutions, to a method of inhibiting the transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor, to a method of bleaching of lignin-containing material, in particular bleaching of pulp for paper production, to a method of treatment of waste water from pulp manufacturing, and to a method of enzymatic polymerization and/or modification of lignin or lignin containing material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/DK93/00395 filed Dec. 1, 1993, which isincorporated herein by reference.

TECHNICAL FIELD

This invention relates to activation of enzymes. More specifically, theinvention relates to agents capable of enhancing the activity ofperoxidases or peroxidase acting compounds.

The invention also relates to methods of oxidizing a substrate with asource of hydrogen peroxide in the presence of a peroxidase enzyme or aperoxidase acting compound, and an enhancing agent. More specifically,the invention relates to a method of bleaching of dye in solutions, to amethod of inhibiting the transfer of a textile dye from a dyed fabric toanother fabric when said fabrics are washed together in a wash liquor,to a method of bleaching lignin-containing material, in particularbleaching of pulp for paper production, to a method of treatment ofwaste water from pulp manufacturing, and to a method of enzymaticpolymerization and/or modification of lignin or lignin containingmaterial.

BACKGROUND ART

Peroxidases (E.C. 1.11.1.7) are enzymes that catalyse the oxidation of asubstrate (an electron or hydrogen donor) with hydrogen peroxide. Suchenzymes are known from microbial, plant and animal origins, e.g.peroxidase from Coprinus cinereus (cf. e.g. EP 179,486). They aretypically hemoproteins, i.e. they contain a heme as a prosthetic group.

Use of peroxidase together with hydrogen peroxide or a hydrogen peroxideprecursor has been suggested e.g. in bleaching of pulp for paperproduction, in treatment of waste water from pulp production, forimproved bleaching in laundry detergents, for dye transfer inhibitionduring laundering, and for lignin modification, e.g. in particle boardproduction.

The compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), ABTS,supplied by Boehringer Mannheim, is a chromogenic substrate, and acommon peroxidase and phenol oxidase assay agent. These enzymes catalysethe oxidation of ABTS by hydrogen peroxide and dioxygen, respectively,producing a greenish-blue colour, which process may be monitoredphotometrically.

ABTS has been found to form a stable radical cation when oxidized by alaccase enzyme (polyphenol oxidase, EC 1.10.3.2), and has been proposedto act as a redox mediator for oxidation of non-phenolic lignin modelcompounds [Bourbonnais R. Paice M G; FEBS Lett (1990) 267 99-102].

Studies on demethylation and delignification of kraft pulp by a laccaseenzyme in the presence of ABTS showed that the extent of partialdemethylation by laccase was increased in the presence of ABTS[Bourbonnais, R. and Paice, M. G; Appl. Microbiol. Biotechnol. (1992) 36823-827].

Certain oxidizable substrates e.g. metal ions and phenolic compoundssuch as 7-hydroxycoumarin (7HCm), vanillin (VAN), andp-hydroxybenzenesulfonate (pHBS), have been described as accelerators orenhancers, able to enhance bleaching reactions (cf. e.g. WO 92/18683, WO92/18687, and Kato M and Shimizu S, Plant Cell Physiol. 1985 26 (7), pp.1291-1301 (cf. Table 1 in particular), or Saunders B C, et al.,Peroxidase, London, 1964, p. 141 ff).

SUMMARY OF THE INVENTION

It has now surprisingly been found that organic chemical compoundsconsisting of at least two aromatic rings, of which aromatic rings atleast one ring is substituted with one or more of the following atoms:nitrogen, oxygen, and sulfur, and which aromatic rings may furthermorebe fused rings, are capable of enhancing the activity of peroxidases andperoxidase acting compounds.

Accordingly, in its first aspect, the invention provides an agent forenhancing the activity of peroxidases or peroxidase acting compounds,which agent is an organic chemical compound consisting of at least twoaromatic rings, of which aromatic rings at least one ring is substitutedwith one or more of the following atoms: nitrogen, oxygen, and sulfur;and which aromatic rings may furthermore be fused rings.

In a more specific aspect, the invention provides an agent for enhancingthe activity of peroxidases or peroxidase acting compounds, which agentis an organic chemical compound of the general formula I: ##STR1##

in which general formula A represents a single bond, or one of thefollowing groups: (--CR¹¹ ═CR¹⁵ --)_(n), (--NR¹¹ --), (--CR¹¹ ═N--)_(n),(--NR¹¹ --CR¹² ═N--N═CR¹³ --NR¹⁵ --), (--NR¹¹ --CR¹² ═N--N═CR¹⁵ --),(--NR¹¹ --CR ¹² ═N--)_(n), (--CR¹² ═N--NR¹¹ --), (--NR¹¹ --CR¹² ═CR¹³--), (--N═N--), in which groups n represents an integer of from 1 to 6;or A represents carbon, carbonyl, nitrogen, sulfur, oxygen, selenium, orphosphor, which carbon, phosphor and nitrogen may be unsubstituted orsubstituted with a substituent group R¹¹ ;

and in which general formula the substituent groups R¹ -R¹³ and R¹⁵,which may be identical or different, independently represents any of thefollowing radicals: hydrogen, halogen, hydroxy, formyl, carboxy andesters and salts hereof, carbamoyl, sulfo and esters and salts hereof,sulfamoyl, phospho, phosphono, phosphonooxy, sulfandiyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR¹⁴ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R¹⁴ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R¹⁴;

which substituent group R¹⁴ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino, C₁ -C₅ -alkyl,C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: halogen,hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl,sulfo and esters and salts hereof, and sulfamoyl; and which C₁ -C₅-alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl;

or in which general formula R⁵ and R⁶ may together form a group --B--,in which B represents a single bond, the group (--CH═CH--)_(n), or thegroup (--CH═N--)_(n), in which groups n represents an integer of from 1to 6; or B represents carbon, nitrogen, sulfur, oxygen, selenium, ortellurium, which carbon and nitrogen may be unsubstituted or substitutedwith a substituent group R¹⁴ as defined above;

or in which general formula two of the substituent groups R¹ -R¹⁰ maytogether form a group --C--, in which C represents any of the followinggroups: (--CHR¹¹ --N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), in whichgroups n represents an integer of from 2 to 4, and in which groups R¹¹is a substituent group as defined above;

or in which general formula R⁵ and R¹², and/or R⁶ and R¹³, when n in theabove formula being 1, may together form a group --D--, in which Drepresents the groups: (--CHR¹¹ --), (--NR¹¹ --), (--CR¹¹ ═CR¹⁵ --),(--CR¹¹ ═N--), (--N═CR¹¹ --), (--O--), (>C═O) or (--S--), and in whichgroups R¹¹ and R¹⁵ are substituent groups as defined above.

In another specific aspect, the invention provides an agent forenhancing the activity of peroxidases or peroxidase acting compounds,which agent is an organic chemical compound of the general formula II:##STR2##

in which the substituent groups R¹ -R⁸, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR⁹ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R⁹ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R⁹ ;

which substituent group R⁹ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, andamino groups may furthermore be unsubstituted or substituted once ortwice with hydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl mayfurthermore be substituted with one or more of the following radicals:halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and whichC₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl;

or in which general formula two of the substituent groups R¹ -R⁸ maytogether form a group --B--, in which B represents any of the followinggroups: (--N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), (--N═CR⁹ --NR¹⁰ --)or (--N═N--CR⁹ --), in which groups n represents an integer of from 1 to3, R⁹ is a substituent group as defined above and R¹⁰ is defined as R⁹.

In another aspect, the invention provides a method for oxidizing asubstrate with a peroxidase or a peroxidase acting compound, in thepresence of a source of hydrogen peroxide, in the presence of anenhancing agent of the invention.

In a more specific aspect, the invention provides a method for bleachingdye in solutions by treatment with a peroxidase enzyme or a peroxidaseacting compound in the presence of a source of hydrogen peroxide in thepresence of an enhancing agent of the invention.

In another specific aspect, the invention provides a method ofinhibiting the transfer of a textile dye from a dyed fabric to anotherfabric when said fabrics are washed together in a wash liquor, themethod comprising treatment of the wash liquor with a peroxidase orperoxidase acting compound in the presence of a source of hydrogenperoxide and in the presence of an enhancing agent of the invention.

In another aspect, the invention provides a method of bleachinglignin-containing material, in particular bleaching of pulp for paperproduction, the method comprising treatment of the lignin or lignincontaining material with a peroxidase or a peroxidase acting compound inthe presence of a source of hydrogen peroxide and in the presence of anenhancing agent of the invention.

In a further aspect, the invention provides a method of enzymaticpolymerization and/or modification of lignin or lignin containingmaterial, the method comprising treatment of the lignin or lignincontaining material with a peroxidase or a peroxidase acting compound inthe presence of a source of hydrogen peroxide and in the presence of anenhancing agent of the invention.

In a yet further aspect, the invention provides a method of treatment ofwaste water, in particular waste water from pharmaceutical or chemicalindustry, e.g. waste water from dye manufacturing, from textileindustry, or from pulp manufacturing, the method comprising treatment ofthe waste water with a peroxidase or a peroxidase acting compound in thepresence of a source of hydrogen peroxide and in the presence of anenhancing agent of the invention.

In a particular aspect, the invention provides a detergent additivecapable of inhibiting the transfer of a textile dye from a dyed fabricto another fabric when said fabrics are washed together in a washliquor, the detergent additive comprising an enzyme exhibitingperoxidase activity or a peroxidase acting compound, a source ofhydrogen peroxide and an enhancing agent of the invention.

In other aspects, the invention provides detergent additives anddetergent compositions capable of inhibiting the transfer of a textiledye from a dyed fabric to another fabric when said fabrics are washedtogether in a wash liquor, the detergent composition comprising anenzyme exhibiting peroxidase activity or a peroxidase acting compound, asource of hydrogen peroxide, and an enhancing agent of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 shows the specific formulas of some enhancing agents of theinvention (I) 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS);(II) 6-hydroxy-2-naphtoic acid; (III) 6-bromo-2-naphtol; (IV)7-methoxy-2-naphtol; (V) 7-amino-2-naphthalene sulfonic acid; (VI)5-amino-2-naphthalene sulfonic acid; (VII) 1,5-diaminonaphthalene;(VIII) 7-hydroxy-1,2-naphthimidazole; (IX) 10-methylphenothiazine; (X)10-phenothiazine-propionic acid; (XI) N-hydroxysuccinimide-10-phenothiazine-propionate; (XII) benzidine; (XIII)3,3'-dimethylbenzidine; (XIV) 3,3'-dimethoxybenzidine; (XV)3,3',5,5'-tetramethylbenzidine; (XVI) 4'-hydroxy-4-biphenylcarboxylicacid; (XVII) 4-amino-4'-methoxystilbene; (XVIII)4,4'-diaminostilbene-2,2'-disulfonic acid; (XIX)4,4'-diaminodiphenylamine, (XX) 2,7-diaminofluorene; (XXI)4,4'-dihydroxy-biphenylene; (XXII) triphenylamine); (XXIII)10-ethyl-4-phenothiazinecarboxylic acid; (XXIV) 10-ethylphenothiazine;(XXV) 10-propylphenothiazine; (XXVI) 10-isopropylphenothiazine; (XXVII)methyl-10-phenothiazinepropionate; (XXVIII) 10-phenylphenothiazine;(XXIX) 10-allylphenothiazine; (XXX) 2-chloro-10-methylphenothiazine;(XXXI) 10-(3-(4-methyl-1-piperazinyl)propyl)phenothiazine; (XXXII)10-(2-pyrrolidinoethyl)phenothiazine; (XXXIII) chlorpromazine; (XXXIV)10-methylphenoxazine; (XXXVI) 6-amino-3-methyl-2-benzothiazolinone azinewith 3-methyl-2-benzothiazolinone; (XXXVII) iminostilbene; (XXXVIII)2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid; (XXXIX)N-benzylidene-4-biphenylamine; (XL) 4,4'-diaminodiphenylaminesulfate;(XLI) 5-amino-2-naphthalenesulfonic acid; (XLII) 1,6-dibromo-2-naphtol;(XLIII) 7-methoxy-2-naphtol; (XLIV) 4,4'-dihydroxybenzophenone; (XLV)N-(4-(dimethylamino)benzylidene)p-anisidine; (XLVI)3-methyl-2-benzothiazolinone(4-(dimethylamino)benzylidene)hydrazone;(XLVII) 2-acethyl-10-methylphenothiazine.

FIG. 2 shows a comparison of an enhancing agent of the invention (ABTS)and pHBS, applied to bleaching of Methyl Orange by a Coprinus cinereusperoxidase (1: pHBS, 20 μM H₂ O₂ ; 2: pHBS, 200 μM H₂ O₂ ; 3: ABTS, 20μM H₂ O₂ ; 4: ABTS, 200 μM H₂ O₂);

FIG. 3 shows accelerated bleaching of Methyl Orange by a Coprinuscinereus peroxidase in the presence of varying concentrations of anenhancing agent of the invention (ABTS) (1: 0 μM ABTS; 2: 1 μM ABTS; 3:5 μM ABTS; and 4: 10 μM ABTS);

FIG. 4 shows a comparison of the initial bleaching rates duringbleaching of Direct Blue 1 (DB1) at pH 10.5 (□ ABTS, 1 nM peroxidase; ♦VAN, 100 nM peroxidase; ▪ 7HCm, 100 nM peroxidase; ▴ pHBS, 100 nMperoxidase); and

FIG. 5 shows a comparison of the initial bleaching rates duringbleaching of DB1 at pH 8.8 (and pH 10.5) (□ ABTS pH 8.8; ♦ VAN pH 8.8; ▪7HCm pH 8.8; ⋄ ABTS pH 10.5; and ▴ pHBS pH 8.8).

FIG. 6 shows the initial bleaching of DB1 at pH 10.5 under the followingconditions: 100 μM H₂ O₂ ; 25 mM Borate; 0.5 PODU/ml Myxococcusvirescens peroxidase; 0 μM 10-propionic acid phenothiazine (□)/10 μM10-propionic acid phenothiazine (♦), respectively; room temperature.

Determination of peroxidase activity: 1 peroxidase unit (PODU) is theamount of enzyme that catalyzes the conversion of 1 μmol hydrogenperoxide per minute at the following analytical conditions: 0.88 mMhydrogen peroxide, 1.67 mM 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate), 0.1 M phosphate buffer, pH 7.0,incubated at 30° C., photometrically followed at 418 nm.

DETAILED DISCLOSURE OF THE INVENTION The Enhancing Agent

The present invention relates to the use of chemical compounds forenhancing the activity of peroxidase enzymes or peroxidase actingcompounds. Accordingly, the invention provides an agent capable ofenhancing the effect of a peroxidase enzyme or a peroxidase actingcompound, in the following termed enhancing agent.

Contrary to the enhancers known and previously described, the enhancingagents of this invention are capable of acting at alkaline conditions,i.e. at pH above 8. This feature allows the enhancers of the inventionto be implemented into various industrial processes.

The enhancing agent of the invention is an organic chemical compoundconsisting of at least two aromatic rings, of which aromatic rings atleast one ring is substituted with one or more nitrogen, oxygen, and/orsulfur atoms, and which aromatic rings may furthermore be fused rings.

In a more preferred embodiment, the enhancing agent of the invention isan organic chemical compound of the general formula I: ##STR3##

in which general formula A represents a single bond, or one of thefollowing groups: (--CR¹¹ ═CR¹⁵ --)_(n), (--NR¹¹ --), (--CR¹¹ ═N--)_(n),(--NR¹¹ --CR¹² ═N--N═CR¹³ --NR¹⁵ --), (--NR¹¹ --CR¹² ═N--N═CR¹⁵ --),(--NR¹¹ --CR¹² ═N--)_(n), (--CR¹² ═N--NR¹¹ --), (--NR¹¹ --CR¹² ═CR¹³--), (--N═N--), in which groups n represents an integer of from 1 to 6;or A represents carbon, carbonyl, nitrogen, sulfur, oxygen, selenium, orphosphor, which carbon, phosphor and nitrogen may be unsubstituted orsubstituted with a substituent group R¹¹ ;

and in which general formula the substituent groups R¹ -R¹³ and R¹⁵,which may be identical or different, independently represents any of thefollowing radicals: hydrogen, halogen, hydroxy, formyl, carboxy andesters and salts hereof, carbamoyl, sulfo and esters and salts hereof,sulfamoyl, phospho, phosphono, phosphonooxy, sulfandiyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR¹⁴ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R¹⁴ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R¹⁴;

which substituent group R¹⁴ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino, C₁ -C₅ -alkyl,C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: halogen,hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl,sulfo and esters and salts hereof, and sulfamoyl; and which C₁ -C₅-alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl;

or in which general formula R⁵ and R⁶ may together form a group --B--,in which B represents a single bond, the group (--CH═CH--)_(n) or thegroup (--CH═N--)_(n), in which groups n represents an integer of from 1to 6; or B represents carbon, nitrogen, sulfur, oxygen, selenium, ortellurium, which carbon and nitrogen may be unsubstituted or substitutedwith a substituent group R¹⁴ as defined above;

or in which general formula two of the substituent groups R¹ -R¹⁰ maytogether form a group --C--, in which C represents any of the followinggroups: (--CHR¹¹ --N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), in whichgroups n represents an integer of from 2 to 4, and in which groups R¹¹is a substituent group as defined above;

or in which general formula R⁵ and R¹², and/or R⁶ and R¹³, when n in theabove formula being 1, may together form a group --D--, in which Drepresents the groups: (--CHR¹¹ --), (--NR¹¹ --), (--CR¹¹ ═CR¹⁵ --),(--CR¹¹ ═N--), (--N═CR¹¹ --), (--O--), (>C═O) or (--S--), and in whichgroups R¹¹ and R¹⁵ are substituent groups as defined above.

In particular embodiments, the enhancing agent is2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate),6-amino-3-methyl-2-benzothiazolinone azine with3-methyl-2-benzothiazolinone,2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid,N-(4-(dimethylamino)benzylidene)-p-anisidine,3-methyl-2-benzothiazolinone(4-(dimethylamino)benzylidene)hydrazone.

In another preferred embodiment, the enhancing agent of the invention isan organic chemical compound of the general formula II: ##STR4##

in which the substituent groups R¹ -R⁸, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR⁹ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R⁹ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R⁹ ;

which substituent group R⁹ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, andamino groups may furthermore be unsubstituted or substituted once ortwice with hydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl mayfurthermore be substituted with one or more of the following radicals:halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and whichC₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl;

or in which general formula two of the substituent groups R¹ -R⁸ maytogether form a group --B--, in which B represents any of the followinggroups: (--N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), (--N═CR⁹ --NR¹⁰ --)or (--N═N--CR⁹ --), in which groups n represents an integer of from 1 to3, R⁹ is a substituent group as defined above and R¹⁰ is defined as R⁹.

In a more specific embodiment, the enhancing agent of the invention isan organic chemical compound of the following formula: ##STR5##

in which formula E represents a single bond, a carbonyl group or one ofthe following groups: (--CH═CH--)_(n), (--CH═N--)_(n) or (--NR¹¹ --), inwhich n represents an integer from 1 to 2. The substituents groups R¹-R¹¹ may be identical or different, independently being one of thefollowing radicals: hydrogen, halogen, hydroxy, formyl, carboxy andesters and salts hereof, carbamoyl, sulfo and esters and salts hereof,sulfamoyl, nitro, amino, phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy,carbonyl-C₁ -C₅ -alkyl, aryl-C₁ -C₅ -alkyl; which carbamoyl, sulfamoyl,and amino groups may furthermore be unsubstituted or substituted once ortwice with a substituent group R¹⁴ ; and which phenyl may furthermore beunsubstituted or substituted with one or more substituent groups R¹⁴ ;and which C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, andaryl-C₁ -C₅ -alkyl groups may be saturated or unsaturated, branched orunbranched, and may furthermore be unsubstituted or substituted with oneor more substituent groups R¹⁴ ;

which substituent group R¹⁴ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, andamino groups may furthermore be unsubstituted or substituted once ortwice with hydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl mayfurthermore be substituted with one or more of the following radicals:halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and whichC₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl;

or in which specific formula two of the substituent groups R⁵ and R⁶ maytogether form a group --B--, in which B represents the groups:(--CH═N--)_(n), (--CH═CH--) or (--CHR¹⁴ --) in which groups n representsan integer of from 1 to 2 and R¹⁴ is a substituent group as definedabove.

In particular embodiments, the enhancing agent is4-amino-4'-methoxystilbene, 4,4'-diaminostilbene-2,2'-disulfonic acid,iminostilbene, 4,4'-dihydroxybenzophenone,N-benzylidene-4-biphenylamine, 4,4'-diaminodiphenylamine,4,4'-diaminodiphenylaminesulfate, 2,7-diaminofluorene, triphenylamine.

In another specific embodiment, the enhancing agent may be described bythe following formula: ##STR6##

in which formula X represents one of the following groups: (--O--),(--S--), (--NR¹⁵ --), (--CHR¹⁵ --), (>C═O), (--CH═CH--), (--CH═N--) andthe substituent groups R¹ -R⁹ and R¹⁵, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR¹⁰ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R¹⁰ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R¹⁰;

which substituent group R¹⁰ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino, C₁ -C₅ -alkyl,C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, and amino groups mayfurthermore be unsubstituted or substituted once or twice with hydroxy,C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; and which phenyl may furthermore besubstituted with one or more of the following radicals: halogen,hydroxy, amino, formyl, carboxy and esters and salts hereof, carbamoyl,sulfo and esters and salts hereof, and sulfamoyl; and which C₁ -C₅-alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl;

or in which general formula two of the substituent groups R¹ -R⁸ maytogether form a group --B--, in which B represents any of the followingthe groups: (--CHR¹⁰ --N═N--), (--CH═CH--)_(n), (--CH═N--)_(n) or(--N═CR¹⁰ --NR¹¹ --), in which groups n represents an integer of from 1to 3, R¹⁰ is a substituent group as defined above and R¹¹ is defined asR¹⁰.

In particular embodiments, the enhancing agent is10-methylphenothiazine, 10-phenothiazine-propionic acid,N-hydroxysuccinimide-10-phenothiazine-propionate or10-ethyl-4-phenothiazine-carboxylic acid, 10-ethylphenothiazine,10-propylphenothiazine, 10-isopropylphenothiazine,methyl-10-phenothiazinepropionate,10-phenylphenothiazine,10-allylphenothiazine,10-(3-(4-methyl-1-piperazinyl)propyl)phenothiazine,10-(2-pyrrolidinoethyl)phenothiazine, chlorpromazine,2-chloro-10-methylphenothiazine, 2-acetyl-10-methylphenothiazine,4-carboxy-10-phenothiazine, 10-methylphenoxazine, 10-ethylphenoxazine,10-phenoxazine-propionic acid or 4-carboxy-10-phenoxazine-propionicacid.

In another specific embodiment, enhancing agent is a biphenyl derivativeof the following formula: ##STR7##

in which the substituent groups R¹ -R¹⁰, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR¹¹ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R¹¹ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R¹¹;

which substituent group R¹¹ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, andamino groups may furthermore be unsubstituted or substituted once ortwice with hydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl mayfurthermore be substituted with one or more of the following radicals:halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and whichC₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl.

In particular embodiments, the enhancing agent is benzidine,3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine,3,3',5,5'-tetramethylbenzidine, 4'-hydroxy-4-biphenylcarboxylic acid, or4,4'-dihydroxybiphenylene.

In another specific embodiment, the enhancing agent is a naphthalenederivative of the following formula: ##STR8##

in which the substituent groups R¹ -R⁸, which may be identical ordifferent, independently represents any of the following radicals:hydrogen, halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; which carbamoyl, sulfamoyl, and amino groups may furthermorebe unsubstituted or substituted once or twice with a substituent groupR⁹ ; and which phenyl may furthermore be unsubstituted or substitutedwith one or more substituent groups R⁹ ; and which C₁ -C₁₄ -alkyl, C₁-C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkyl groups maybe saturated or unsaturated, branched or unbranched, and may furthermorebe unsubstituted or substituted with one or more substituent groups R⁹ ;

which substituent group R⁹ represents any of the following radicals:halogen, hydroxy, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, sulfamoyl, nitro, amino,phenyl, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; which carbamoyl, sulfamoyl, andamino groups may furthermore be unsubstituted or substituted once ortwice with hydroxy, C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy; and which phenyl mayfurthermore be substituted with one or more of the following radicals:halogen, hydroxy, amino, formyl, carboxy and esters and salts hereof,carbamoyl, sulfo and esters and salts hereof, and sulfamoyl; and whichC₁ -C₅ -alkyl, and C₁ -C₅ -alkoxy groups may furthermore be saturated orunsaturated, branched or unbranched, and may furthermore be substitutedonce or twice with any of the following radicals: halogen, hydroxy,amino, formyl, carboxy and esters and salts hereof, carbamoyl, sulfo andesters and salts hereof, and sulfamoyl;

or in which general formula two of the substituent groups R¹ -R⁸ maytogether form a group --B--, in which B represents any of the followinggroups: (--N═N--), (--CH═CH--)_(n), (--CH═N--)_(n), (--N═CR⁹ --NR¹⁰ --)or (--N═N--CR⁹ --), in which groups n represents an integer of from 1 to3, R⁹ is a substituent group as defined above and R¹⁰ is defined as R⁹.

In particular embodiments, the enhancing agent is 6-hydroxy-2-naphtoicacid, 6-bromo-2-naphtol, 7-methoxy-2-naphtol, 7-amino-2-naphthalenesulfonic acid, 5-amino-2-naphthalene sulfonic acid,1,5-diaminonaphthalene, 7-hydroxy-1,2-naphthimidazole,5-amino-2-naphthalenesulfonic acid, 1,6-dibromo-2-naphtol or7-methoxy-2-naphtol.

The enhancing agent of the invention may be in free form or in the formof an addition salt.

Methods of Oxidizing a Substrate

In another aspect, the invention provides a method of oxidizing asubstrate with a source of hydrogen peroxide in the presence of aperoxidase enzyme or a peroxidase acting compound, in the presence of anenhancing agent of the invention.

The enhancing agent of the invention may be present in free form or inthe form of an addition salt.

The enhancing agent of the invention may be present in concentrations offrom 0.01 to 500 μM, more preferred 0.1 to 250 μM, most preferred 1 to100 μM.

The source of hydrogen peroxide may be hydrogen peroxide or a hydrogenperoxide precursor for in situ production of hydrogen peroxide, e.g.percarbonate or perborate, or a hydrogen peroxide generating enzymesystem, e.g. an oxidase and a substrate for the oxidase or an amino acidoxidase and a suitable amino acid, or a peroxycarboxylic acid or a saltthereof. Hydrogen peroxide may be added at the beginning or during theprocess, e.g. in an amount of 0.001-5 mM, particularly 0.01-1 mM.

Peroxidases and Peroxidase Acting Compounds

The enzyme employed in the method of the invention may be any peroxidaseenzyme comprised by the enzyme classification EC 1.11.1.7, or anyfragment derived therefrom, exhibiting peroxidase activity, or syntheticor semisynthetic derivatives thereof (e.g. porphyrin ring systems ormicroperoxidases, cf. e.g. U.S. Pat. No. 4,077,768, EP PatentApplication 537,381, International Patent Applications WO 91/05858 andWO 92/16634). Such enzymes are known from microbial, plant and animalorigins.

Preferably, the peroxidase employed in the method of the invention isproducible by plants (e.g. horseradish or soy bean peroxidase) ormicroorganisms such as fungi or bacteria. Some preferred fungi includestrains belonging to the subdivision Deuteromycotina, classHyphomycetes, e.g. Fusarium, Humicola, Tricoderma, Myrothecium,Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia,Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672),Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113),Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERMP-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli orDreschlera halodes.

Other preferred fungi include strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g. Coprinus, Phanerochaete,Coriolus or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.NA-12) or Trametes (previously called Polyporus), e.g. T. versicolor(e.g. PR4 28-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(IFO 12382) or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Bacillus pumilus (ATCC 12905), Bacillusstearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri,Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonasfluorescens (NRRL B-11).

Further preferred bacteria include strains belonging to Myxococcus, e.g.M. virescens.

Other potential sources of useful particular peroxidases are listed inSaunders B C, op. cit., pp. 41-43.

The peroxidase may furthermore be one which is producible by a methodcomprising cultivating a host cell transformed with a recombinant DNAvector which carries a DNA sequence encoding said peroxidase as well asDNA sequences encoding functions permitting the expression of the DNAsequence encoding the peroxidase, in a culture medium under conditionspermitting the expression of the peroxidase and recovering theperoxidase from the culture.

Particularly, a recombinantly produced peroxidase is a peroxidasederived from a Coprinus sp., in particular C. macrorhizus or C. cinereusaccording to WO 92/16634.

In the context of this invention, peroxidase acting compounds compriseperoxidase active fragments derived from cytochromes, hemoglobin orperoxidase enzymes, and synthetic or semisynthetic derivatives thereof,e.g. iron porphins, iron porphyrins, and iron phthalocyanine andderivatives thereof.

INDUSTRIAL APPLICATIONS

Due to their excellent performance at alkaline conditions, the enhancingagents of the invention, and hence the method for oxidizing a substratewith a peroxidase enzyme or a peroxidase acting compound in the presenceof a source of hydrogen peroxide and in the presence of an enhancing ofthe invention, find various industrial applications

In a preferred embodiment, the method of the invention finds applicationfor bleaching of dye in solutions.

In another embodiment, the method of the invention finds application fordye transfer inhibition, e.g. for treatment of dyed textiles (cf. e.g.WO 92/18687) or during laundering (cf. e.g. WO 91/05839).

Accordingly, in a specific embodiment, the invention provides a methodfor inhibiting the transfer of a textile dye from a dyed fabric toanother fabric when said fabrics are washed together in a wash liquor,the method comprising treatment of the wash liquor with a peroxidaseenzyme or a peroxidase acting compound in the presence of a source ofhydrogen peroxide, and in the presence of an enhancing agent of theinvention. The textile dye may be a synthetic dye such as an azo dye, ora natural or nature-identical dye.

In a third embodiment, the method of the invention finds application inbleaching of pulp for paper production. The use of a peroxidase togetherwith hydrogen peroxide or a hydrogen peroxide precursor in bleaching ofpaper pulp has been described in e.g. SE 88/0673 and U.S. Pat. No.4,690,895.

Accordingly, the invention provides a method for bleaching oflignin-containing material, in particular bleaching of pulp for paperproduction, which method comprises treatment of the lignin or lignincontaining material with a peroxidase enzyme or a peroxidase actingcompound in the presence of a source of hydrogen peroxide and in thepresence of an enhancing agent of the invention.

In a fourth embodiment, the method of the invention finds applicationfor lignin modification, e.g. in particle board production. Binders forproducing wood composites such as fibre boards and particle boards canbe made from peroxidase treated lignin (cf. U.S. Pat. No. 4,432,921).

Accordingly, the invention provides a method for enzymaticpolymerization and/or modification of lignin or lignin containingmaterial, which method comprises treatment of the lignin or lignincontaining material with a peroxidase or a peroxidase acting compound inthe presence of a source of hydrogen peroxide, and in the presence of anenhancing agent of the invention.

In a fifth embodiment, the method of the invention finds application intreatment of waste water e.g. waste water from the chemical orpharmaceutical industry, from dye manufacturing, from dye-works, fromthe textile industry, or from pulp production (cf. e.g. U.S. Pat. No.4,623,465, or JP-A-2-31887).

In a more specific aspect, the invention provides a method for treatmentof waste water from dye manufacturing, from dye-works, from textileindustry, or from pulp manufacturing, the method comprising treatment ofthe waste water with a peroxidase or a peroxidase acting compound in thepresence of a source of hydrogen peroxide and in the presence of anenhancing agent of the invention.

Detergent Compositions

Due to their excellent performance at alkaline conditions the enhancingagents of the invention are particularly well suited for implementationinto detergent compositions, intended for performance in the range of pH7-13, particularly the range of pH 8-12, preferably the range of pH8-11.

According to the invention, the enhancing agent may be added as acomponent of a detergent composition. As such, it may be included in thedetergent composition in the form of a detergent additive. The detergentcomposition as well as the detergent additive may additionally compriseone or more other enzymes conventionally used in detergents, such asproteases, lipases, amylases, oxidases, and cellulases.

In a specific aspect, the invention provides a detergent additive. Theenzymes may be included in a detergent composition by adding separateadditives containing one or more enzymes, or by adding a combinedadditive comprising all of these enzymes. A detergent additive of theinvention, i.e. a separated additive or a combined additive, can beformulated e.g. as granulates, liquids, slurries, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids,slurries, or protected enzymes.

Dust free granulates may be produced, e.g. as disclosed in U.S. Pat. No.4,106,991 and U.S. Pat. No. 4,661,452, and may optionally be coated bymethods known in the art. The detergent enzymes may be mixed before orafter granulation.

Liquid enzyme preparations may, for instance, be stabilized usingconventional stabilizing agents for the enzymes, as described below.

Protected enzymes may be prepared according to the method disclosed inEP 238,216 A.

In another specific aspect, the invention provides a detergentcomposition capable of inhibiting the transfer of textile dyes from dyedfabrics to other fabrics when said fabrics are washed together in a washliquor, the detergent composition comprising an enzyme or compoundexhibiting peroxidase activity, a source of hydrogen peroxide and anenhancing agent of the invention.

The enhancing agent of the invention may be included in the detergentcomposition as a part of a peroxidase system, comprising one or moreperoxidase enzymes or peroxidase acting compounds, a source of hydrogenperoxide, and the enhancing agent of the invention.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules or liquid. A liquid detergent may beaqueous, typically containing up to 70% water and 0-20% organic solvent.

The detergent composition comprises one or more surfactants each ofwhich may be anionic, non-ionic, cationic or amphoteric. The detergentwill usually contain 5-30% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(AS), alcohol ethoxysulfate (AES) or soap. It may also contain 3-20% ofnon-ionic surfactant such as nonylphenol ethoxylate or alcoholethoxylate.

The detergent composition may additionally comprise one or more otherenzymes, such as an amylase, lipase, cellulase or protease.

The detergent may contain 1-40% of a detergent builder such as zeolite,phosphate, phosphonate, citrate, nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaaceticacid (DTPA), alkenylsuccinic anhydride, or silicate, or it may beunbuilt (i.e. essentially free of a detergent builder). It may alsocontain other conventional detergent ingredients, e.g. fabricconditioners, foam boosters, anti-corrosion agents, soil-suspendingagents, sequestering agents, anti-soil redeposition agents, stabilizingagents for the enzyme(s), foam depressors, dyes, bactericides, opticalbrighteners or perfumes.

The pH (measured in aqueous detergent solution) will usually be neutralor alkaline, e.g. 7-11.

Particular forms of detergent composition within the scope of theinvention include:

a) A detergent composition formulated as a detergent powder containingphosphate builder, anionic surfactant, nonionic surfactant, silicate,alkali to adjust to desired pH in use, and neutral inorganic salt.

b) A detergent composition formulated as a detergent powder containingzeolite builder, anionic surfactant, nonionic surfactant, acrylic orequivalent polymer, silicate, alkali to adjust to desired pH in use, andneutral inorganic salt.

c) A detergent composition formulated as an aqueous detergent liquidcomprising anionic surfactant, nonionic surfactant, organic acid,alkali, with a pH in use adjusted to a value between 7 and 11.

d) A detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant consisting essentially of linearalkoxylated primary alcohol, phosphate builder, alkali, with a pH in useadjusted to a value between about 7 and 11.

e) A compact detergent composition formulated as a detergent powder inthe form of a granulate having a bulk density of at least 600 g/l,containing anionic surfactant and nonionic surfactant, phosphatebuilder, silicate, and little or substantially no neutral inorganicsalt.

f) A compact detergent composition formulated as a detergent powder inthe form of a granulate having a bulk density of at least 600 g/l,containing anionic surfactant and nonionic surfactant, zeolite builder,silicate, and little or substantially no neutral inorganic salt.

g) A detergent composition formulated as a detergent powder containinganionic surfactant, nonionic surfactant, acrylic polymer, fatty acidsoap, carbonate, sulfate, clay particles, and silicate.

h) A liquid compact detergent comprising 5-65% by weight of surfactant,0-50% by weight of builder and 0-30% by weight of electrolyte.

i) A compact granular detergent comprising linear alkylbenzenesulfonate,tallow alkyl sulfate, C₁₄₋₁₅ alkyl sulfate, C₁₄₋₁₅ alcohol 7 timesethoxylated, tallow alcohol 11 times ethoxylated, dispersant, siliconefluid, trisodium citrate, citric acid, zeolite, maleic acid/acrylic acidcopolymer, diethylenetriaminepentakis (methylenephosphonic acid),cellulase, protease, lipase, amylase, sodium silicate, sodium sulfate,PVP, perborate and bleach activator.

j) A granular detergent comprising sodium linear C₁₁₋₁₂alkylbenzenesulfonate, sodium sulfate, zeolite A, sodiumnitrilotriacetate, cellulase, PVP, tetraacetylethylenediamine, boricacid and perborate.

k) A liquid detergent comprising C₁₂₋₁₄ alkenylsuccinic acid, citricacid, sodium C₁₂₋₁₅ alkyl sulfate, sodium sulfate of C₁₂₋₁₅ alcohol 2times ethoxylated, C₁₂₋₁₅ alcohol 7 times ethoxylated, C₁₂₋₁₅ alcohol 5times ethoxylated, diethylenetriaminepentakis(methylenephosphonic acid),oleic acid, ethanol, propanediol, protease, cellulase, PVP, sudssupressor, sodium hydroxide, perborate and bleach activator.

The following examples further illustrate the present invention, andthey are not intended to be in any way limiting to the scope of theinvention as claimed.

EXAMPLE 1 Bleaching of Methyl Orange

Coprinus cinereus peroxidase (CiP) was obtained according to WO92/16634, and purified to a single band on SDS-PAGE by the followingmethod:

The crude peroxidase preparation was precipitated with 25% w/wanmoniumsulfate, and after centrifugation the precipitate was dissolvedin 10 mM phosfate pH 7 (buffer A) and dialysed against the same buffer.The sample was applied onto a HighLoad Q-Sepharose column (Pharmacia,Sweden) equilibrated with buffer A, washed with buffer and eluted with alinear gradient of NaCl up to 0.5 M in the same buffer.

The main fraction containing peroxidase activity was collected,concentrated by ultrafiltration (with a membrane cut-off of 10 kD) anddialysed against buffer A.

The concentration of CiP was determined by A₄₀₄ using a molar absorptionof 109 mM⁻¹ cm⁻¹.

Accelerated bleaching of Methyl orange (Merck) catalysed by CiP andhydrogen peroxide in the presence of2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS, supplied byBoehringer Mannheim) or para-hydroxybenzene sulfonate (pHBS, supplied bySigma) is shown in FIG. 2. The following conditions were used:

10 nM CiP

25 μM Methyl Orange

50 μM ABTS or para-hydroxybenzene sulfonate

20 or 200 μM hydrogen peroxide

50 mM Britton & Robinson buffer*, pH 8.8

30° C. thermostat

Reagents were mixed in a 1 cm cuvette, and the bleaching was started byaddition of hydrogen peroxide. The bleaching was detectedspectrophotometrically at 465 nm, which is the absorption peak of thisdye. Bleaching was followed with respect to time over a span of 10 min.

EXAMPLE 2 Bleaching of Methyl Orange

Accelerated bleaching of Methyl Orange (Merck) catalysed by a Coprinuscinereus peroxidase (CiP), obtained according to Example 1, and hydrogenperoxide in the presence of varying concentrations of2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS, supplied byBoehringer Mannheim) is shown in FIG. 3. The following conditions wereused:

10 nM CiP

25 μM Methyl Orange

0, 1, 5 or 10 μM ABTS

200 μM hydrogen peroxide

50 μM Britton & Robinson buffer, pH 8.8

30° C. thermostat

Mixture, start and detection of the bleaching are as described inExample 1.

EXAMPLE 3 Bleaching of Direct Blue 1

The initial bleaching of Direct Blue 1 (DB1) by a Coprinus cinereusperoxidase (CiP), obtained according to Example 1, using a selection ofenhancers according to the invention was compared to the known enhancer,p-hydroxybenzene sulfonate, sodium salt (pHBS). Chemicals were obtainedfrom Sigma-Aldrich, Janssen Chimica, Kodak, Tokyo Kasai OrganicChemicals, Daiichi Pure Chemicals Co. or Boehringer Mannheim, and someN-methylated derivatives of phenothiazine were made by methylation withmethyliodide as described by Cornel Bodea and Ioan Silberg in "RecentAdvances in the Chemistry of Phenothiazines" (Advances in heterocyclicchemistry, 1968, Vol. 9, pp. 321-460); B. Cardillo & G. Casnati inTetrahedron, 1967, Vol. 23, p. 3771.

Due to low solubility some of the enhancers were dissolved in a smallvolume of ethanol before dilution in water.

The following conditions were used:

    ______________________________________                                                             Final concentration                                      ______________________________________                                        200 μl 50 mM Britton-Robinson buffer,                                                           10       mM                                                pH 8.5 and 10.5, respectively,                                                200 μl DB1 ˜ 3.0 Abs. Units (610 nm) 0.6 (A.sub.610nm)                                            200 μl 50 nM CiP in water 10 nM                                            200 μl 50 μM enhancer 10 μM                                          200 μl 100 μM H.sub.2 O.sub.2 20                                       μM                                           ______________________________________                                    

Reagents were mixed in a 1 cm thermostated cuvette at 30° C. and thebleaching was started by addition of hydrogen peroxide.

The bleaching was detected spectrophotometrically at 610 nm, which isthe absorption peak of DB1. After 5 sec. bleaching was followed for 4minutes, and the initial bleaching rates (reduction in milli-absorbanceunits per minute, -ΔmAbs/min, determined from the initial slope of theabsorbance curve after 5 sec.) as well as the total bleaching within 4minutes (100×(A_(610nm),start -A_(610nm),4min)/A_(610nm),start %) weredetermined.

A_(610nm),start was determined by replacement of hydrogen peroxide withwater.

From the results presented in Table 1, below, it appears that theenhancers of the invention are superior to the known enhancer, pHBS, ininitial bleaching of DB1, especially at high pH values.

Determination of the bleaching after 4 minutes is carried out to verifythat a reasonable total bleaching can be obtained, and data are due topossible hydrogen peroxide deficiency not directly comparable.

                  TABLE 1                                                         ______________________________________                                        Bleaching of Direct Blue 1 at pH 8.5 and pH 10.5                                              Initial DB1 bleaching                                            (-ΔmAbs/min) DB1 bleaching in 4 min.                                 Enhancer    pH 8.5   pH 10.5  pH 8.5 pH 10.5                                  ______________________________________                                        ABTS        1044     197      86%    86%                                        10-Phenothiazine- 1080 468  85% 88%                                           propionic acid                                                                10-methyl- 1176 480  85% 87%                                                  phenothiazine                                                                 4'-hydroxy- 588 68 57% 39%                                                    4-biphenyl-                                                                   carboxylic acid                                                               3,3',5,5'- 960 266  63% 49%                                                   tetramethyl-                                                                  benzidine                                                                     4,4'- 222 61 58% 34%                                                          diaminostilbene-                                                              2,2'-disulfonic                                                               acid                                                                          4-amino-4'- 132 72 46% 26%                                                    methoxy-stilbene                                                              1,5-diamino- 240 90 37% 32%                                                   naphthalene                                                                   6-hydroxy-2- 486 52 46% 32%                                                   naphtoic acid                                                                 10-ethyl-4-pheno- 1146 864  85% 89%                                           thiazinecarboxylic                                                            acid                                                                          10-ethylpheno- 1098 624  83% 84%                                              thiazine                                                                      10-propylpheno- 1068 299  80% 81%                                             thiazine                                                                      10-isopropylpheno- 681 59 83% 41%                                             thiazine                                                                      methyl-10-pheno- 840 99 86% 67%                                               thiazinepropionate                                                            10-phenylpheno- 498 40 81% 25%                                                thiazine                                                                      10-allylpheno- 1170 183  86% 80%                                              thiazine                                                                      N-hydroxysuccin- 1110 378  84% 75%                                            imide-10-phenothi-                                                            azinepropionic acid                                                           10-(3-(4-methyl-1- 1092 237  87% 82%                                          piperazinyl)propyl)                                                           phenothiazine                                                                 10-(2-pyrrolidino- 444 160  86% 80%                                           ethyl)phenothia-                                                              zine                                                                          Chlorpromazine 222 26 61% 16%                                                 10-methylphenoxa- 1464 792  79% 81%                                           zine                                                                          6-amino-3-methyl-2- 96 42 54% 29%                                             benzothiazolinone                                                             azine w. 3-methyl-2-                                                          benzothiazolinone                                                             iminostilbene 186 56 35% 21%                                                  2-(p-aminophenyl)- 114 26 25% 11%                                             6-methylbenzothia-                                                            zole-7-sulfonic acid                                                          N-benzylidene- 474 38 66% 28%                                                 4-biphenylamine                                                               4,4'-diaminodiphe- 378 28 59%  8%                                             nylaminesulfate                                                               5-amino-2-naphtha- 816 63 71% 26%                                             lenesulfonic acid                                                             1,6-dibromo-2-naph- 222 58 70% 23%                                            tol                                                                           7-methoxy-2-naphtol 480 84 46% 29%                                            4,4'-dihydroxyben- 238 10 81%  4%                                             zophenone                                                                     N-(4-(dimethylami- 294 24 49%  9%                                             no)benzylidene)-p-                                                            anisidine                                                                     3-methyl-2-benzo- 378 26 66%  8%                                              thiazolinone(4-(di-                                                           methylamino)ben-                                                              zylidene)hydrazone                                                            2,7-diamino- 636  516 39% 49%                                                 fluorene,2HCl                                                                 2-chloro-10- 225 15 86%  8%                                                   methylpheno-                                                                  thiazine                                                                      2-acetyl-10- 270 25 88% 18%                                                   methylpheno-                                                                  thiazine                                                                      pHBS 57 ˜0 42% ˜0%                                                No enhancer 25 ˜0 18% ˜0%                                       ______________________________________                                    

EXAMPLE 4 Bleaching of Direct Blue 1 with ABTS

The initial bleaching of Direct Blue 1 (DB1) by a Coprinus cinereusperoxidase (CiP), obtained according to Example 1, using ABTS asaccelerator, was compared to the best of the hitherto knownaccelerators: 7-hydroxycoumarin (7HCm), vanillin (VAN), andp-hydroxybenzene sulfonate (pHBS). The following conditions were used:

1 nM CiP or 100 nM CiP (at pH 10.5)

0, 10, 25, 50, or 75 μM accelerator, respectively

50 mM Britton & Robinson buffer, pH 8.8 or 10.5, respectively

20 μM hydrogen peroxide

Reagents were mixed in a 1 cm cuvette, and the bleaching was started byaddition of hydrogen peroxide. The bleaching was detectedspectrophotometrically at 610 nm, which is the absorption peak of thisdye. Bleaching was followed for 10 minutes, and the bleaching rates(-ΔmAbs/min) were determined from the initial (linear) reduction inabsorbance.

At pH 10.5 the bleaching using 100 nm CiP and ABTS as accelerator was sofast that bleaching was already completed before the cuvette could beplaced in the spectrophotometer, the reason why the dosage of CiP at pH10.5 was reduced to 1 nM when used in combination with ABTS, although adosage near 100 nM CiP was necessary for all other (hitherto known)accelerators in order to see a significant reduction in absorbance.

The results of initial bleaching rate per minute have been illustratedin FIGS. 4 and 5 as function of accelerator concentration.

EXAMPLE 5 Enhanced Dye Transfer Inhibition by ABTS

A washing trial was carried out in a Terg-o-tometer to investigate theeffect of ABTS on peroxidase based dye transfer inhibition. For acomparison, also the established enhancer pHBS was tested.

Clean white tracer test pieces (cotton, Style#400 from Testfabrics,Inc., U.S.A.; bleached, but unbrightened) were washed together withnylon test pieces dyed with the azo dye Acid Red 151 (C.I. 26900;available, e.g. from Aldrich Chemical Co.). Reference test pieces werecut out of the same cotton cloth and washed in the absence of dyedfabric. The dye transfer in a given Terg-o-tometer pot was measured asthe Hunter colour difference ##EQU1## between the tracer pieces in thatpot and the above reference pieces, the Hunter L, a, and b values beingevaluated from remission data obtained with an unfiltered daylightsource on a Datacolor Elrephometer 2000.

The detergent solution for the washing trial was made up using 4.5 g/lof a commercially available European high-pH powder detergent containingno bleach and no optical brightener. The water used was tap water mixedwith demineralized water in the ratio 1:2; the mixture had a hardnessequivalent to approx. 1.1 mM Ca²⁺.

The detailed experimental conditions were:

Duration of wash: 15 min.

Terg-o-tometer agitation: 70 rotations/min.

Temperature: 35° C.

pH: Adjusted to 10.5 with NaOH prior to addition of peroxidase system

Textile load: Approx. 6 g nylon dyed with acid Red 151 and 1 g whitecotton per liter washing liquor

Peroxide source: In all cases, 50 μM H₂ O₂ was present together with theperoxidase

Peroxidase: Coprinus cinereus peroxidase obtained according to Example 1at 5 nM

After washing, the test pieces were rinsed thoroughly in cold tap waterand dried in the dark overnight, after which the remission measurementswere performed.

Treatments with various concentrations of the two enhancers yielded thefollowing results:

    ______________________________________                                                     Hunter ΔE with respect to                                     white, washed fabric                                                       ______________________________________                                         1 μM ABTS                                                                              34.9                                                                5 μM ABTS 32.3                                                            20 μM ABTS 23.7                                                             1 μM pHBS 34.8                                                             5 μM pHBS 34.5                                                            20 μM pHBS 30.8                                                          ______________________________________                                    

Differences of ≧2 units of Hunter ΔE were statistically significant.

In both cases, the peroxidase system with 1 μM enhancer provided nosignificant dye transfer inhibition (reference without peroxidase systemnot included here). However, as is seen that the ABTS system has aneffect already at 5 μM of enhancer, whereas the pHBS system does not;and at 20 μM enhancer, the ABTS system has a much larger effect than thepHBS system.

EXAMPLE 6 Bleaching of Direct Blue 1

A crude soy bean peroxidase (SBP) was purified by anion and cationchromatography followed by gelfiltration to a single protein on SDS-PAGEwith an R₂ -value (A_(404nm) /A_(280nm)) of 2.2:

125 ml of crude SBP were adjusted to pH 7, diluted to 2.3 mS andfiltered through 0.8μ filter. The sample was applied to 300 ml of DEAEcolumn equilibrated with 20 mM phosphate pH 7.0, and the peroxide waseluted with a 1 M NaCl linear gradient in the same buffer. Fractionswith peroxidase activity were pooled.

Pooled fractions from anion exchange chromatography (190 ml) wereconcentrated and washed by ultrafiltration (GR61PP) membrane from Dow,Denmark). pH was adjusted to 5.3 ionic strength to 2.3 mS in the samplebefore application to a 200 ml S-Sepharose column previouslyequilibrated with 50 mM acetate pH 5.3. The effluent containing theperoxidase activity was concentrated and washed by ultrafiltration to afinal volume of approx. 10 ml.

A 5 ml concentrated sample from cation exchange chromatography wasapplied to a 90 cm Sephacryl S-200 column equilibrated and eluted with0.1 M acetate pH 6.1.

Fractions with peroxidase activity giving only one band on SDS-PAGE werepooled.

The bleaching rate of direct blue 1 (DB1) by the purified SBP wasdetermined using a selection of enhancers according to the invention.The following conditions were used:

    ______________________________________                                                            Final concentration                                       ______________________________________                                        200 μl 50 mM Britton-Robinson buffer                                                             10 mM                                                     pH 6, 8 and 10, respectively                                                  200 μl DB1 ˜ 3.0 Abs.Units (610 nm) 0.6 (A.sub.610nm)                200 μl SBP with A.sub.404nm = 0.0005 at pH 6 0.0001 or                     and 8 or with A.sub.404nm = 0.005 at pH 10 0.001 (A.sub.404 nm)                                    200 μl 50 μM enhancer 10 μM                     200 μl 100 μM H.sub.2 O.sub.2 20 μM                                ______________________________________                                    

Reagents were mixed in a thermostated cuvette at 30° C. and thebleaching was started by addition of hydrogen peroxide.

The bleaching was detected and calculated as in Example 3.

From the results presented in Tables 2 and 3 below, it appears that byadding enhancers of the invention we can obtain a much faster bleachingof the dye compared to the experiments without enhancer.

                  TABLE 2                                                         ______________________________________                                        Bleaching of Direct Blue 1 with SBP in 4 Minutes                                                 % DB1 bleaching in 4 min.                                                                   pH 10                                          Enhancer pH 6 pH 8 10x[SBP]                                                 ______________________________________                                        No enhancer      0.7       <0.7    <0.7                                         10-Phenothiazine 72 61 21                                                     propionic acid                                                                10-Ethyl-4-phenothiazine 69 69 32                                             carboxylic acid                                                               10-Methylphenothiazine 67 54 12                                               4'-Hydroxy-4-biphenyl- 61 47 10                                               carboxylic acid                                                               10-Methylphenoxazine 68 67 65                                                 pHBS   <0.7   <0.7   <0.7                                                   ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Initial Bleaching of Direct Blue 1 with SBP                                                      (-ΔmAbs/min)                                                                          pH 10                                          Enhancer pH 6 pH 8 10x[SBP]                                                 ______________________________________                                        No enhancer     <1         <1    <1                                             10-Phenothiazine 162  84 33                                                   propionic acid                                                                10-Ethyl-4-phenothiazine 228 120 45                                           carboxylic acid                                                               10-Methylphenothiazine 204 102 30                                             4'-Hydroxy-4-biphenyl- 237 132  9                                             carboxylic acid                                                               10-Methylphenoxazine 258 180 89                                               pHBS  <1  <1 <1                                                             ______________________________________                                    

EXAMPLE 7

Myxococcus virescens, DSM 8593, was inoculated on "5 CY" agar plateswith the following composition:

    ______________________________________                                               Casitone        3 g                                                      Yeast extract 1 g                                                             CaCl.2H.sub.2 O 1 g                                                           Agar 15 g                                                                          Water ad 1000 ml, pH 7.2                                               ______________________________________                                    

and incubated for 2 weeks at 26° C.

The agar was cut into pieces and transferred aseptically to 5 shakeflasks with 100 ml "MD-1M"-medium in each flask.

The flasks were incubated on a rotary shaker at 250 rpm, 26° C., for 5days. The combined culture from the 5 flasks was used for inoculating 50flasks with 100 ml "MD-1M"-medium in each, using a 10 ml inoculum perflask.

The 50 flasks were incubated on a rotary shaker at 250 rpm, 26° C., for3 days. The peroxidase activity as described above was measured in thecombined culture from the 50 flasks, result: 0.2 PODU/ml.

After separation of the solid material by centrifugation the peroxidasewas concentrated by ultrafiltration using a 10 kDa cut off membrane. Theultrafiltrated preparation had an activity of 2.9 PODU/ml.

The ultrafiltrated preparation described above was used in a dyebleaching experiment with Direct Blue 1 (DB1) at pH 10.5 (FIG. 6), withand without an enhancer of the invention, using the followingconditions:

100 μM H₂ O₂

25 mM Borate pH 10.5

0.5 PODU/ml Myxococcus virescens peroxidase

0 or 10 μM 10-phenothiazinepropionic acid, respectively, roomtemperature

the initial concentration of DB1 was adjusted to give an OD_(610nm)=0.6.

Reagents were mixed in a 1 cm cuvette, and the bleaching was started byaddition of hydrogenperoxide. The bleaching was detectedspectrophotometrically at 610 nm for 120 seconds.

FIG. 6 shows that the effect of the enhancer is very pronounced. It alsoshows that the absorbance is reduced to at least half the initial valueafter 30 sec. in the experiment with 10 μM 10-propionic acidphenothiazine.

EXAMPLE 8 Bleaching of Direct Blue 1

Horse radish peroxidase type VI (HRPC) was obtained from Sigma(8P-8375).

The bleaching rate of direct blue 1 (DB1) by HRPC was determined using aselection of enhancers according to the invention. The followingconditions were used:

    ______________________________________                                                            Final concentration                                       ______________________________________                                        200 μl 50 mM Britton-Robinson buffer                                                             10 mM                                                     pH 8 and 10, respectively                                                     200 μl DB1 ˜ 3.0 Abs.Units (610 nm) 0.6 (A.sub.610nm)                200 μl HRPC with A.sub.404nm = 0.005 0.001 (A.sub.404nm)                   200 μl 50 μM enhancer 10 μM                                          200 μl 100 μM H.sub.2 O.sub.2 20 μM                                ______________________________________                                    

Reagents were mixed in a thermostated cuvette at 30° C., and thebleaching was started by addition of hydrogen peroxide.

The bleaching was detected and calculated as in Example 3.

From the results presented in Table 4 below, it appears that by addingenhancers of the invention we can obtain a much faster bleaching of thedye compared to the experiment without enhancer.

                  TABLE 4                                                         ______________________________________                                        Initial Bleaching of Direct Blue 1 with HRPC                                                          -ΔmAbS/min                                      Enhancer            pH 8   pH 10                                              ______________________________________                                        No enhancer          <1    <1                                                   10-Phenothiazine 1061 62                                                      propionic acid                                                                10-Ethyl-4-phenothiazine 1038 68                                              carboxylic acid                                                               10-Methylphenothiazine 1164 83                                                10-Methylphenoxazine 1188 99                                                  pHBS  <2 <2                                                                 ______________________________________                                         ##SPC1##

We claim:
 1. A detergent composition comprising(a) a surfactant, (b) anenzyme exhibiting peroxidase activity, (c) one or more other enzymesselected from the group consisting of protease, lipase, amylase,cellulase and oxidase, and (d) an enhancing agent of formula I ##STR9##wherein X is --O-- or --S--; R¹ -R⁹ independently are hydrogen, halogen,hydroxy, formyl, carboxy or an ester or salt thereof, carbamoyl, sulfoor an ester or salt thereof, sulfamoyl, phospho, phosphono, phosphonooxyor an ester or salt thereof, amino, phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅-alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁ -C₅ -alkyl; wherein thecarbamoyl, sulfamoyl, and amino are unsubstituted or substituted once ortwice with R¹⁰ ; the phenyl is unsubstituted or substituted with one ormore R¹⁰ ; and the C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅-alkyl, and aryl-C₁ -C₅ -alkyl are saturated or unsaturated, branched orunbranched, and unsubstituted or substituted with one or more R¹⁰ ;wherein R¹⁰ is halogen, hydroxy, formyl, carboxy or an ester or saltthereof, carbamoyl, sulfo or an ester or salt thereof, sulfamoyl,phospho, phosphono, phosphonooxy or an ester or salt thereof, nitro,amino, phenyl, aminoalkyl, piperidino, piperazinyl, pyrrolidino, C₁ -C₅-alkyl, C₁ -C₅ -alkoxy; wherein the carbamoyl, sulfamoyl, and amino areunsubstituted or substituted once or twice with hydroxy, C₁ -C₅ -alkyl,C₁ -C₅ -alkoxy; wherein the phenyl may be substituted with one or moreof halogen, hydroxy, amino, formyl, carboxy or an ester or salt thereof,carbamoyl, sulfo or an ester or salt thereof, and sulfamoyl; and whereinthe C₁ -C₅ -alkyl and C₁ -C₅ -alkoxy are saturated or unsaturated,branched or unbranched, and may be substituted once or twice with any ofhalogen, hydroxy, amino, formyl, carboxy or an ester or salt thereof,carbamoyl, sulfo or an ester or salt thereof, and sulfamoyl; or two ofthe substituent groups R¹ -R⁸ together form a group --B--, wherein B is(--CHR¹⁰ --N═N--), (--CH═CH--)_(n), (--CH═N--)_(n) or (--N═CR¹⁰ --NR¹⁰--) wherein n is 1, 2 or
 3. 2. The detergent composition of claim 1,wherein X is --O--.
 3. The detergent composition of claim 1, wherein Xis --S--.
 4. The detergent composition of claim 2, wherein R¹ -R⁹ areindependently hydrogen, halogen, hydroxy, amino, phenyl, C₁ -C₁₄ -alkyl,C₁ -C₅ -alkoxy, wherein the C₁ -C₁₄ -alkyl and C₁ -C₅ -alkoxy areunsubstituted, saturated or unsaturated, branched or unbranched.
 5. Thedetergent composition of claim 3, wherein R¹ -R⁹ are independentlyhydrogen, halogen, hydroxy, amino, phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅-alkoxy, wherein the C₁ -C₁₄ -alkyl and C₁ -C₅ -alkoxy areunsubstituted, saturated or unsaturated, branched or unbranched.
 6. Thedetergent composition of claim 2, wherein R⁵ is an alkyl group which isunsubstituted or substituted with one or more R¹⁰.
 7. The detergentcomposition of claim 3, wherein R⁵ is an alkyl group which isunsubstituted or substituted with one or more R¹⁰.
 8. The detergentcomposition of claim 4, wherein R⁵ is an alkyl group which isunsubstituted or substituted with one or more R¹⁰.
 9. The detergentcomposition of claim 5, wherein R⁵ is an alkyl group which isunsubstituted or substituted with one or more R¹⁰.
 10. The detergentcomposition of claim 1, wherein the enhancing agent is10-methylphenothiazine, 10-phenothiazine-propionic acid,N-hydroxysuccinimide-10-phenothiazine-propionate,10-ethyl-4-phenothiazine-carboxylic acid, 10-ethylphenothiazine,10-propylphenothiazine, 10-isopropylphenothiazine,methyl-10-phenothiazinepropionate, 10-phenylphenothiazine,10-allylphenothiazine,10-(3-(4-methyl-1-piperazinyl)propyl)phenothiazine,10-(2-pyrrolidinoethyl)phenothiazine,2-chloro-10-methylphenothiazine,2-acetyl-10-methylphenothiazine,4-carboxy-10-phenothiazine-propionicacid or chlorpromazine.
 11. The detergent composition of claim 1,wherein the enhancing agent is 10-methylphenoxazine,10-ethylphenoxazine, 10-phenoxazine-propionic acid or4-carboxy-10-phenoxazine-propionic acid.
 12. The detergent compositionof claim 1, wherein the enzyme exhibiting peroxidase activity ishorseradish peroxidase, soy bean peroxidase or a peroxidase derived fromCoprinus, Bacillus, or Myxococcus.
 13. The detergent composition ofclaim 1, wherein the amount of the enhancing agent is in the range offrom 0.01-500 μM.
 14. The detergent composition of claim 1 which has apH in the range of from 7-13.
 15. The detergent composition of claim 14which has a pH in the range of from 7-11.
 16. The detergent compositionof claim 14 which has a pH in the range of from 8-12.
 17. The detergentcomposition of claim 15 which has a pH in the range of from 8-11.
 18. Amethod of washing a fabric, comprising treating the fabric with(a) asurfactant, (b) an enzyme exhibiting peroxidase activity, and (c) one ormore other enzymes selected from the group consisting of protease,lipase, amylase, cellulase and oxidase, and (d) an enhancing agent offormula I: ##STR10## wherein X is --O-- or --S--; R¹ -R⁹ independentlyare hydrogen, halogen, hydroxy, formyl, carboxy or an ester or saltthereof, carbamoyl, sulfo or an ester or salt thereof, sulfamoyl,phospho, phosphono, phosphonooxy or an ester or salt thereof, amino,phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, aryl-C₁-C₅ -alkyl; wherein the carbamoyl, sulfamoyl, and amino areunsubstituted or substituted once or twice with R¹⁰ ; the phenyl isunsubstituted or substituted with one or more R¹⁰ ; and the C₁ -C₁₄-alkyl, C₁ -C₅ -alkoxy, carbonyl-C₁ -C₅ -alkyl, and aryl-C₁ -C₅ -alkylare saturated or unsaturated, branched or unbranched, and unsubstitutedor substituted with one or more R₁₀ ; wherein R₁₀ is halogen, hydroxy,formyl, carboxy or an ester or salt thereof, carbamoyl, sulfo or anester or salt thereof, sulfamoyl, phospho, phosphono, phosphonooxy or anester or salt thereof, nitro, amino, phenyl, aminoalkyl, piperidino,piperazinyl, pyrrolidino, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; wherein thecarbamoyl, sulfamoyl, and amino are unsubstituted or substituted once ortwice with hydroxy, C₁ -C₅ -alkyl, C₁ -C₅ -alkoxy; wherein the phenylmay be substituted with one or more of halogen, hydroxy, amino, formyl,carboxy or an ester or salt thereof, carbamoyl, sulfo or an ester orsalt thereof, and sulfamoyl; and wherein the C₁ -C₅ -alkyl and C₁ -C₅-alkoxy are saturated or unsaturated, branched or unbranched, and may besubstituted once or twice with any of halogen, hydroxy, amino, formyl,carboxy or an ester or salt thereof, carbamoyl, sulfo or an ester orsalt thereof, and sulfamoyl; or two of the substituent groups R¹ -R⁸together form a group --B--, wherein B is (--CHR¹⁰ --N═N--),(--CH═CH--)_(n), (--CH═N--)_(n) or (--N ═CR¹⁰ --NR¹⁰ --) wherein n is 1,2 or
 3. 19. The method of claim 18, wherein X is --O--.
 20. The methodof claim 18, wherein X is --S--.
 21. The method of claim 19, wherein R¹-R⁹ are independently hydrogen, halogen, hydroxy, amino, phenyl, C₁ -C₁₄-alkyl, C₁ -C₅ -alkoxy, wherein the C₁ -C₁₄ -alkyl and C₁ -C₅ -alkoxyare unsubstituted, saturated or unsaturated, branched or unbranched. 22.The method of claim 20, wherein R¹ -R⁹ are independently hydrogen,halogen, hydroxy, amino, phenyl, C₁ -C₁₄ -alkyl, C₁ -C₅ -alkoxy, whereinthe C₁ -C₁₄ -alkyl and C₁ -C₅ -alkoxy are unsubstituted, saturated orunsaturated, branched or unbranched.
 23. The method of claim 19, whereinR⁵ is an alkyl group which is unsubstituted or substituted with one ormore R¹⁰.
 24. The method of claim 20, wherein R⁵ is an alkyl group whichis unsubstituted or substituted with one or more R¹⁰.
 25. The method ofclaim 21, wherein R⁵ is an alkyl group which is unsubstituted orsubstituted with one or more R¹⁰.
 26. The method of claim 22, wherein R⁵is an alkyl group which is unsubstituted or substituted with one or moreR¹⁰.
 27. The method of claim 18, wherein the enhancing agent is10-methylphenothiazine, 10-phenothiazine-propionic acid,N-hydroxysuccinimide-10-phenothiazine-propionate,10-ethyl-4-phenothiazine-carboxylic acid, 10-ethylphenothiazine,10-propylphenothiazine, 10-isopropylphenothiazine,methyl-10-phenothiazinepropionate, 10-phenylphenothiazine,10-allylphenothiazine,10-(3-(4-methyl-1-piperazinyl)propyl)phenothiazine,10-(2-pyrrolidinoethyl)phenothiazine,2-chloro-10-methylphenothiazine,2-acetyl-10-methylphenothiazine,4-carboxy-10-phenothiazine-propionicacid or chlorpromazine.
 28. The method of claim 18, wherein theenhancing agent is 10-methylphenoxazine, 10-ethylphenoxazine,10-phenoxazine-propionic acid or 4-carboxy-10-phenoxazine-propionicacid.
 29. The method of claim 18, wherein the enzyme exhibitingperoxidase activity is horseradish peroxidase, soy bean peroxidase or aperoxidase derived from Coprinus, Bacillus, or Myxococcus.
 30. Themethod of claim 18, wherein the amount of the enhancing agent is in therange of from 0.01-500 μM.
 31. The method of claim 18, wherein thefabric is treated at a pH in the range of from 7-13.
 32. The method ofclaim 31 which the fabric is treated at a pH in the range of from 7-11.33. The method of claim 31 which the fabric is treated at a pH in therange of from 8-12.
 34. The method of claim 32 which the fabric istreated at a pH in the range of from 8-11.